US20230320370A1 - Method of producing processed protein - Google Patents

Method of producing processed protein Download PDF

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US20230320370A1
US20230320370A1 US18/044,453 US202118044453A US2023320370A1 US 20230320370 A1 US20230320370 A1 US 20230320370A1 US 202118044453 A US202118044453 A US 202118044453A US 2023320370 A1 US2023320370 A1 US 2023320370A1
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protein
transglutaminase
laccase
tofu
processed
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Kiyota Sakai
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Amano Enzyme Inc
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Amano Enzyme Inc
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A23C11/106—Addition of, or treatment with, microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/16—Vegetable proteins from soybean
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/40—Pulse curds
    • A23L11/45—Soy bean curds, e.g. tofu
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A23L11/65—Soy drinks
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

  • the present invention relates to a method of producing a processed protein. More specifically, the present invention relates to a processing technique for enhancing the crosslinking effect of a protein.
  • a protein is crosslinked for a predetermined purpose. For example, for the purpose of improving an existing processed food product, creating a processed food product having new preference characteristics, and the like, a crosslinking treatment is performed on a protein as a food product material.
  • Non-Patent Document 1 As a means used for crosslinking a protein, a transglutaminase is known.
  • the transglutaminase crosslinks a side chain carbamoyl group of a glutamine residue and a side chain amino group of a lysine residue of a protein with an isopeptide bond.
  • Patent Documents 1 and 2 describe that the strength of tofu is enhanced by using a transglutaminase in production of tofu.
  • Patent Document 3 describes that a protein is crosslinked by a laccase, and further describes that a laccase crosslinks a protein by forming an s-amino group of lysine with another amino group to form a Schiff base.
  • an object of the present invention is to provide a processing technique for enhancing the crosslinking effect of a protein.
  • the present inventor has found that the crosslinking effect of a protein is remarkably enhanced by combining a laccase and a transglutaminase.
  • the crosslinking effect to be obtained is generally poor and that both of a laccase and a transglutaminase has the same side chain amino acid of lysine as a substrate, it has been unexpected that the crosslinking effect of the protein is remarkably enhanced by a combination of these enzymes.
  • the unexpected effect obtained by such a combination has been remarkable to the extent that the crosslinking effect of the protein is remarkably enhanced by further combining a transglutaminase even when a large amount of laccase is used so that the crosslinking effect of the protein is almost saturated.
  • the present inventor has conducted further studies based on the findings, leading to the completion of the present invention. That is, the present invention provides inventions of the following aspects.
  • FIG. 1 shows the relationship between the presence or absence of a laccase treatment and a difference in concentration of a laccase in production of tofu, and the strength of tofu, as obtained in a preliminary test example.
  • FIG. 2 shows the relationship between the presence or absence of a laccase treatment, the presence or absence of a transglutaminase treatment, and a difference in concentration of a transglutaminase in production of tofu, and the strength of tofu, as obtained in Test Example 1.
  • a method of producing a processed protein of the present invention includes a crosslinking step of causing a laccase and a transglutaminase to act on a protein.
  • the method of producing a processed protein will be specifically described.
  • a supply source of the protein used in the present invention is not particularly limited, and a material containing a protein is used without particular limitation.
  • the material containing a protein include materials used in various industrial fields such as food product materials, medical materials, and industrial materials.
  • Specific examples of the protein include plant proteins and animal proteins.
  • the plant proteins include bean proteins such as soybean protein and horse bean protein; and grain proteins such as wheat protein, rye protein, oat protein, and corn protein.
  • the animal proteins include fish protein, livestock protein, egg protein, milk protein, and tendon protein (such as gelatin and collagen).
  • proteins may be used singly or in combination of a plurality of kinds thereof.
  • plant proteins are preferable, bean proteins are more preferable, and soybean protein is particularly preferable.
  • the protein-containing material may be in a form in which a protein and an enzyme are efficiently in contact with each other, and preferably, in the case of an animal protein, examples of the form thereof include minced meat or ground meat of animal food materials (such as fish meat and livestock meat); egg liquids such as whole liquid egg, egg white liquid, and egg yolk liquid; and animal milk such as cow milk and goat milk; and in the case of a plant protein, examples of the form thereof include plant milk, typically, squeeze (milk) of water-absorbed products of food materials (beans such as soybeans and horse beans; grains such as wheat, rye, oats, corn; and the like), and bean powder and grain powder.
  • a purified protein can be used as the protein-containing material.
  • protein-containing materials may be used singly or in combination of a plurality of kinds thereof.
  • plant milk is preferable and soy milk is more preferably preferable.
  • the amount of the protein in the plant milk is not particularly limited, and is, for example, 3 g/100 mL or more, preferably 3.8 g/100 mL or more, more preferably 4.2 g/100 mL or more, further preferably 4.8 g/100 mL or more, and particularly preferably 5 g/100 mL or more.
  • the upper limit of the protein amount range in the plant milk is not particularly limited, and is, for example, 8 g/100 mL or less.
  • the material solid content of the plant milk (for example, the soybean solid content in the case of soy milk) is not particularly limited, and is, for example, 6 wt % or more, preferably 8 wt % or more, more preferably 9 wt % or more, further preferably 9.5 wt % or more, and particularly preferably 10 wt % or more.
  • the upper limit of the material solid content range of the plant milk is not particularly limited, and is, for example, 18 wt % or less or 16 wt % or less.
  • the protein-containing material may contain a quality improving agent such as an emulsifier (such as sucrose fatty acid ester, phospholipid, monoglyceric fatty acid ester, organic acid monoglyceric fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyglycerin fatty acid ester, or propylene glycol fatty acid ester) and a pH adjusting agent (such as sodium carbonate, sodium hydrogen carbonate, or calcium carbonate), a coloring agent, a flavor, a seasoning (such as dietary salt or sugar (sucrose)), or the like according to the form of a target processed protein, and the like, as long as the effect of the present invention is not impaired.
  • an emulsifier such as sucrose fatty acid ester, phospholipid, monoglyceric fatty acid ester, organic acid monoglyceric fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid
  • the laccase used in the present invention is an enzyme having phenol oxidase activity (EC1.10.3.2).
  • specific examples of the laccase include laccases derived from microorganisms such as fungi and bacteria, and more specific examples thereof include laccases derived from the genera Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Pycnoporus, Pyricularia, Trametes, Rhizoctonia, Rigidoporus, Coprinus, Psatyrella, Myceliophtera, Schtalidium, Polyporus, Phlebia, Coriolus , and the like.
  • laccases may be used singly or in combination of a plurality of kinds thereof.
  • a laccase derived from the genus Trametes is preferable, and a laccase derived from Trametes hirsuta is particularly preferable, from the viewpoint of further enhancing the crosslinking effect of the protein.
  • the use amount of the laccase is not particularly limited, but the use amount of the laccase per 1 g of the protein is, for example, 0.02 U or more. From the viewpoint of further enhancing the crosslinking effect of the protein, the use amount of the laccase per 1 g of the protein is preferably 0.2 U or more, more preferably 2 U or more, further preferably 10 U or more, even more preferably 30 U or more, and further more preferably 45 U or more.
  • the upper limit of the use amount range of the laccase is not particularly limited, but the use amount of the laccase per 1 g of the protein is, for example, 500 U or less, 200 U or less, 100 U or less, 80 U or less, 60 U or less, or 55 U or less.
  • the laccase activity when a 0.1 ml enzyme liquid is added to 3 ml of a 1.0 mg/ml solution of 2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) as a substrate at 25° C., the amount of the enzyme that increases the absorbance at 405 nm by 1.0 OD per minute is defined as 1 unit.
  • ABTS 2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate
  • the transglutaminase used in the present invention is an enzyme having transglutaminase activity (EC2.3.2.13).
  • the transglutaminase both a calcium-dependent transglutaminase, which requires calcium for active expression, and a calcium-independent transglutaminase, which does not require calcium for active expression, are mentioned.
  • Specific examples of the transglutaminase include transglutaminases derived from microorganisms, mammals, fish, and the like, and more specific examples thereof include transglutaminases derived from microorganisms belonging to the genera. Streptomyces, Bacillus, Geobacillus , and the like; mammals such as guinea pig (liver), cow (blood), and pig (blood); and fish such as salmon, red sea bream, and cod.
  • transglutaminases may be used singly or in combination of a plurality of kinds thereof.
  • a calcium-independent transglutaminase is preferable.
  • a microorganism-derived transglutaminase is preferable, a transglutaminase derived from the genus Streptomyces is more preferable, and a transglutaminase derived from Streptomyces mobaraensis is particularly preferable.
  • the use amount of the transglutaminase is not particularly limited, but the use amount of the transglutaminase per 1 g of the protein is, for example, 0.001 U or more, 0.01 U or more, or 0.02 U or more. From the viewpoint of further enhancing the crosslinking effect of the protein, the use amount of the transglutaminase per 1 g of the protein is preferably 0.2 U or more, more preferably 0.5 U or more, further preferably 1 U or more, even more preferably 1.5 U or more, and further more preferably 2 U or more, 5 U or more, 10 U or more, or 15 U or more.
  • the upper limit of the use amount range of the transglutaminase is not particularly limited, but the use amount of the transglutaminase per 1 g of the protein is, for example, 200 U or less, 100 U or less, 50 U or less, 30 U or less, 25 U or less, or 20 U or less.
  • the amount of the enzyme that produces 1 ⁇ mol of hydroxamic acid per minute is defined as 1 unit.
  • the ratio of the use amounts of the laccase and the transglutaminase is determined depending on the use amount of each enzyme, but from the viewpoint of further enhancing the crosslinking effect of the protein, the use amount of the transglutaminase with respect to 1 U of the laccase is preferably 0.00001 U or more, 0.0001 U or more, or 0.0004 U or more, more preferably 0.004 U or more, further preferably 0.01 U or more, even more preferably 0.02 U or more, further more preferably 0.03 U or more, and particularly preferably 0.04 U or more, 0.1 U or more, 0.2 U or more, or 0.3 U or more.
  • the upper limit of the use amount range of the transglutaminase with respect to 1 U of the laccase is not particularly limited, and is, for example, 10 U or less, 5 U or less, 2 U or less, 1 U or less, 0.6 U or less, 0.5 U or less, or 0.4 U or less.
  • a protein composition containing a protein-containing material, a laccase, and a transglutaminase is prepared by mixing a protein-containing material, a laccase, and a transglutaminase, typically, adding a laccase and a transglutaminase to a protein-containing material, and the protein composition is maintained in a heated state, thereby allowing the crosslinking of the protein by the laccase and the transglutaminase to proceed.
  • the temperature of the protein-containing material at the time of preparing the protein composition is not particularly limited, and the material may be in a non-heated state or in a heated state, but is preferably in a heated state.
  • the temperature when the material is in a heated state is not particularly limited, but is preferably a temperature for allowing crosslinking described below to proceed.
  • a treatment according to the form of a target processed protein may be simultaneously performed.
  • a coagulation reaction may be performed simultaneously
  • the form of the processed protein is a fermented product such as fermented soy milk (yogurt such as soy milk yogurt, and the like)
  • fermentation may be performed simultaneously.
  • the protein composition to be subjected to the crosslinking step can contain other materials necessary for obtaining the form of a target processed protein, in addition to the protein-containing material, the laccase, and the transglutaminase.
  • the other materials include coagulants used for the form of tofu, and fermenting bacteria (such as yogurt inoculums) used for the form of a fermented product such as fermented soy milk (yogurt such as soy milk yogurt, and the like).
  • the coagulant is not particularly limited, and a coagulant generally used in production of tofu can be used.
  • examples of the coagulant include a salt coagulant and an acid coagulant.
  • examples of the salt coagulant include magnesium chloride, magnesium sulfate, calcium sulfate, and calcium chloride, and examples of the acid coagulant include glucono delta-lactone.
  • coagulants may be used singly or in combination of a plurality of kinds thereof.
  • the coagulant is preferably a salt coagulant and more preferably magnesium chloride.
  • the use amount of the coagulant is not particularly limited, and is, for example, 0.1 wt % or more, 0.2 wt % or more, 0.3 wt % or more, or 0.5 wt % or more. From the viewpoint of further enhancing the crosslinking effect of the protein, the use amount of the coagulant is preferably 0.75 wt % or more, more preferably 1 wt % or more, further preferably 1.5 wt % or more, and even more preferably 2 wt % or more.
  • the upper limit of the use amount range of the coagulant is not particularly limited, and is, for example, 4 wt % or less or 3 wt % or less.
  • the fermenting bacteria such as yogurt inoculums are not particularly limited, and can be appropriately determined in consideration of an optimum temperature of a laccase and a transglutaminase, and the like.
  • the yogurt inoculums include thermophilic bacterial species having an optimum temperature for growth of about 37° C. to 45° C. and mesophilic bacterial species having an optimum temperature for growth of 20° C. to 30° C., and any bacterial species can be used. That is, even when the crosslinking step is performed under heating conditions, not only thermophilic bacterial species but also mesophilic bacterial species can be used.
  • the thermophilic bacterial species include bacterial species belonging to the genera Streptococcus, Enterococcus , and the like.
  • mesophilic bacterial species include bacterial species belonging to the genera Lactococcus (preferably Lactococcus lactis subsp. cremoris ), Lactobacillus, Acetobacter (preferably Acetobacter orientalis ), and the like.
  • yogurt inoculums may be used singly or in combination of two or more kinds thereof.
  • bacterial species belonging to the genus Lactococcus and bacterial species belonging to the genera Lactobacillus and Acetobacter are preferable, Lactococcus lactis subsp. cremoris and Acetobacter orientalis are more preferable, and a combination of these yogurt inoculums is further preferable.
  • the temperature at which the protein composition is provided for allowing crosslinking to proceed can be appropriately determined depending on an optimum temperature of a laccase and a transglutaminase, the form of a target processed protein, and the like, and is, for example, 35 to 70° C., preferably 38 to 60° C., and more preferably 40 to 57° C. More specifically, when the form of a target processed protein is tofu, the temperature for allowing crosslinking to proceed can be appropriately determined depending on an optimum temperature of a laccase and a transglutaminase, and the like, and is, for example, 50 to 70° C., preferably 52 to 60° C., and more preferably 54 to 57° C.
  • the temperature for allowing crosslinking to proceed can be appropriately determined depending on an optimum temperature of a laccase and a transglutaminase, an optimum temperature for growth of fermenting bacteria (such as yogurt bacterial species), and the like, and is, for example, 35 to 50° C., preferably 38 to 46° C., and more preferably 40 to 44° C.
  • the time for crosslinking is not particularly limited, and is, for example, 30 minutes or longer and preferably 1 hour or longer, although it depends on a treatment to be simultaneously performed in the crosslinking step (for example, a coagulation treatment when the form of a processed protein is tofu, a fermentation treatment when the form of a processed protein is a fermented product such as fermented soy milk, and the like), the scale of the protein composition, and the like.
  • the upper limit of the time range for crosslinking is not particularly limited, and is, for example, 30 hours or shorter, 24 hours or shorter, 12 hours or shorter, 8 hours or shorter, or 4 hours or shorter.
  • an arbitrary treatment suitable for the form of a processed protein can be appropriately performed.
  • the arbitrary treatment include a boiling step, a firing (roasting, toasting, baking, grilling, broiling) step, a steaming step, and a frying step. These steps may be used singly or in combination of a plurality of kinds thereof.
  • a specific form of a processed protein (that is, a crosslinked protein) obtained by the production method of the present invention is determined according to the type of the protein, the type of the protein-containing material, and the crosslinking effect to be obtained, and examples thereof include tofu such as cotton tofu, silken tofu, and packed tofu; fermented products such as fermented soy milk and fermented animal milk (yogurt such as soy milk yogurt and animal milk yogurt, cheese, and the like); other soybean processed products such as soybean meat, deep-fried tofu, and tofu hamburger steak; fish paste products such as fish balls, fish rings, minced and steamed fish, fish sausages, and minced fish; livestock processed products such as hamburger steak, sausage, meat dumpling, and minced meat cutlet; egg processed products such as rolled egg, egg filling, scrambled egg, tinned egg, omelet sheet, and long egg; grain processed products such as noodles, confectionery, and bakery; viscoelastic confectionery such as jelly, gummies
  • tofu fermented soy milk (preferably soy milk yogurt), and other soybean processed products are preferable, tofu and other soybean processed products are more preferable, and tofu is further preferable.
  • the crosslinking effect to be obtained of a processed protein by the production method of the present invention is not particularly limited as long as it has a characteristic of being changed by crosslinking of the protein, and examples thereof include improvement of organization characteristics such as enhancement of compressive strength, enhancement of viscoelasticity, and improvement of gel forming ability.
  • the enzyme activity of the laccase was measured by the method described below using 2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS, manufactured by Boehringer Mannheim) as a substrate.
  • ABTS 2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate
  • ABTS was dissolved in a 25 mM citrate buffer solution (pH 3.2) at a concentration of 1.0 mg/ml to prepare a substrate solution.
  • a substrate solution In a cuvette, 3.0 ml of this substrate solution was placed and preheated at 25° C., a 0.1 ml enzyme liquid was then added, stirred, and incubated at 25° C., and the absorbance at 405 nm after 1 minute and 3 minutes was measured. The amount of the enzyme that increased the absorbance at 405 nm by 1.0 OD per minute under this condition was defined as 1 unit (U).
  • a substrate solution was prepared by dissolving 2.42 g of 2-amino-2-hydroxymethyl-1.3-propanediol, 0.70 g of hydroxyammonium hydrochloride, 0.31 g of reduced glutathione, and 1.01 g of Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) in distilled water to make a total amount of 100 mL (pH 6.0).
  • a coloring solution was prepared by mixing 30 mL of a 3 M hydrochloric acid solution, 30 mL of a 12 wt % trichloroacetic acid solution, and 30 mL of 5 wt % iron(III) chloride solution.
  • An enzyme was diluted with 200 nM of Tris-HCl (pH 6.0) to an appropriate concentration to prepare a sample solution. After 100 ⁇ L of the substrate solution was added to 10 ⁇ L. of the sample solution and mixed, the mixture was reacted at 37° C. for 10 minutes. After 100 ⁇ L of the coloring solution was added to stop the reaction and form an Fe complex, the absorbance at 525 nm was measured. As a control, the previously heat-inactivated sample solution was used and reacted in the same manner, the absorbance of the sample solution was measured, and the absorbance difference from the non-inactivated sample solution was determined.
  • a soy milk composition was prepared by heating 20 mL of soy milk at 55° C. for 5 minutes, adding 30 U, 60 U, or 120 U of a laccase aqueous solution per 1 g of the protein and mixing the mixture for 5 seconds or adding 0.6 mL (final concentration: 1.0 wt %) of a 10 wt % magnesium chloride aqueous solution, and mixing the mixture for 5 seconds, tofu was prepared by heating the soy milk composition at 55° C. for 1 hour, and the obtained tofu was cooled at 4° C.
  • a soy milk composition was prepared by heating 20 mL of soy milk at 55° C. for 5 minutes, adding a laccase aqueous solution so as to be the amount shown in Table 1 per 1 g of the protein, mixing the mixture for 5 seconds, adding a transglutaminase aqueous solution so as to be the amount shown in Table 1 per 1 g of the protein, mixing the mixture for 5 seconds, adding 0.6 mL (final concentration: 1.0 wt %) of a 10 wt % magnesium chloride aqueous solution, and mixing the mixture for 5 seconds, tofu was prepared by heating the soy milk composition at 55° C. for 1 hour (crosslinking step associated with coagulation), and the obtained tofu was cooled at 4° C. (Comparative Examples 1-1 to 1-6 and Examples 1-1 to 1-4).
  • the strength (Firmness (N); specifically, compressive strength) of the obtained tofu was measured using a rheometer (COMPAC-100II SUN SCIENTIFIC CO., LTD.). The measurement conditions were as follows: Mode: 20, adapter: No. 13, repetition: 1 time, and pushing distance: 5 mm. Results are shown in FIG. 2 .
  • the strength was enhanced to 3.0 times (specifically, increased by 39 N) when the addition amount of the transglutaminase per 1 g of soybean protein was 0.02 U (Comparative Example 1-2), the strength was enhanced to 5.4 times (specifically, increased by 88 N) when the addition amount of the transglutaminase per 1 g of soybean protein was 0.2 U (Comparative Example 1-3), the strength was enhanced to 8 times (specifically, increased by 140 N) when the addition amount of the transglutaminase per 1 g of soybean protein was 2 U (Comparative Example 1-4), and the strength was enhanced to 8.3 times (specifically, increased by 145 N) when the addition amount of the transglutaminase per 1 g of soybean protein was 20 U (Comparative Example 1-4).
  • the laccase and the transglutaminase have a common substrate (a side chain amino group of a lysine residue), and when the laccase and the transglutaminase coexist, it is reasonably expected that the reaction efficiency of the transglutaminase is reduced by the amount of the substrate lost by the reaction by the laccase.
  • a substrate for a laccase that is different from a transglutaminase (specifically, a tyrosine residue)
  • a protein molecule in which tyrosine residues are crosslinked by the laccase reduces a contact opportunity between the lysine residue and the transglutaminase due to steric hindrance, and it is reasonably expected that the reaction efficiency by the transglutaminase is reduced also in this respect.
  • Sucrose was added to 20 mL of soy milk so as to have a final concentration of 5 wt %, sterilized by autoclaving, cooled to 42° C., and kept warm. While maintaining the temperature at 42° C., 50 U of a laccase per 1 g of soybean protein was added and mixed for 5 seconds, and 20 U of a transglutaminase per 1 g of soybean protein was added and mixed for 5 seconds, yogurt inoculums ( Lactococcus lactis subsp. Cremoris FC and Acetobacter orientalis FA) were added so that the total final concentration was 0.2 wt %, and mixed for 5 seconds, and the mixture was incubated at 42° C.
  • yogurt inoculums Lactococcus lactis subsp. Cremoris FC and Acetobacter orientalis FA
  • soy milk yogurt (Example 2).
  • Soy milk yogurt (Comparative Example 2-1) prepared in the same manner except that neither laccase nor transglutaminase was used, soy milk yogurt (Comparative Example 2-2) prepared in the same manner using only a laccase between a laccase and a transglutaminase, and soy milk yogurt (Comparative Example 2-3) prepared in the same manner using only a transglutaminase between a laccase and a transglutaminase were prepared.
  • the strength (Firmness (N)) of the obtained soy milk yogurt was measured in the same manner as in Test Example 1.

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